The invention relates to a novel protein kinase nucleic acid sequence and protein. Also provided are vectors, host cells, and recombinant methods for making and using the novel molecules.
Phosphate tightly associated with a molecule, e.g., a protein, has been known since the late nineteenth century. Since then, a variety of covalent linkages of phosphate to proteins have been found. The most common involve esterification of phosphate to serine, threonine, and tyrosine with smaller amounts being linked to lysine, arginine, histidine, aspartic acid, glutamic acid, and cysteine. The occurrence of phosphorylated molecules, e.g., proteins, implies the existence of one or more kinases, e.g., protein kinases, capable of phosphorylating various molecules, e.g., amino acid residues on proteins, and also of phosphatases, e.g., protein phosphatases, capable of hydrolyzing various phosphorylated molecules, e.g., phosphorylated amino acid residues on proteins.
Protein kinases play critical roles in the regulation of biochemical and morphological changes associated with cellular growth and division (D""Urso et al. (1990) Science 250:786-791; Birchmeier et al. (1993) Bioessays 15:185-189). They serve as growth factor receptors and signal transducers and have been implicated in cellular transformation and malignancy (Hunter et al. (1992) Cell 70:375-387; Posada et al. (1992) Mol. Biol. Cell 3:583-592; Hunter et al. (1994) Cell 79:573-582). For example, protein kinases have been shown to participate in the transmission of signals from growth-factor receptors (Sturgill et al. (1988) Nature 344:715-718; Gomez et al. (1991) Nature 353:170-173), control of entry of cells into mitosis (Nurse (1990) Nature 344:503-508; Maller (1991) Curr. Opin. Cell Biol. 3:269-275) and regulation of actin bundling (Husain-Chishti et al. (1988) Nature 334:718-721).
Protein kinases can be divided into different groups based on either amino acid sequence similarity or specificity for either serine/threonine or tyrosine residues. A small number of dual-specificity kinases have also been described. Within the broad classification, kinases can be further subdivided into families whose members share a higher degree of catalytic domain amino acid sequence identity and also have similar biochemical properties. Most protein kinase family members also share structural features outside the kinase domain that reflect their particular cellular roles. These include regulatory domains that control kinase activity or interaction with other proteins (Hanks et al. (1988) Science 241:42-52).
Extracellular-signal-regulated kinases/microtubule-associated protein kinases (Erk MAPKs) and cyclin-directed kinases (Cdks) represent two large families of serine-threonine kinases (see Songyang et al., (1996) Mol. Cell. Biol. 16: 6486-6493). Both types of kinases function in cell growth, cell division, and cell differentiation, in response to extracellular stimulae. The Erk MAPK family members are critical participants in intracellular signaling pathways. Upstream activators as well as the Erk MAPK components are phosphorylated following contact of cells with growth factors or hormones or after cellular stressors, for example, heat, ultraviolet light, and inflammatory cytokines. These kinases transport messages that have been relayed from the plasma membrane to the cytoplasm by upstream kinases into the nucleus where they phosphorylate transcription factors and effect gene transcription modulation (Karin et al., (1995) Curr. Biol. 5: 747-757). Substrates of the Erk MAPK family include c-fos, c-jun, APF2, and ETS family members Elk1, Sap1a, and c-Ets-1 (cited in Brott et al., (1998) Proc. Natl Acad. Sci. USA 95, 963-968).
Cdks regulate transitions between successive stages of the cell cycle. The activity of these molecules is controlled by phosphorylation events and by association with cyclin. Cdk activity is negatively regulated by the association of small inhibitory molecules (Dynlacht, (1997) Nature 389:148-152). Cdk targets include various transcriptional activators such as p110Rb, p107 and transcription factors, such as p53, E2F and RNA polymerase II, as well as various cytoskeletal proteins and cytoplasmic signaling proteins (cited in Brott et al., above).
A protein has been isolated in Drosophilia, designated nemo, which has homology to Erk MAPKs and Cdks. A mammalian homologue of nemo, designated NLK, has been reported (Brott et al., above). This protein kinase autophosphorylates and localizes to a great extent in the nucleus. This protein showed homology to both families of kinases (Erk MAPKs and Cdks). It did not possess the characteristic MAPK phosphorylation motif TXY in the conserved kinase domain VIII. It instead exhibited the sequence TQE resembling the THE sequence found in some Cdks.
More recently, it was shown that NLK could down-regulate HMG-domain-containing proteins related to POP-1. The signaling protein Wnt regulates transcription factors containing high-mobility group (HMG) domains to direct decisions on cell fate during animal development. In C. elegans, the HMG-domain-containing repressor POP-1 distinguishes the fate of anterior daughter cells from posterior daughter cells throughout development. Wnt signaling down-regulates POP-1 activity in posterior daughter cells, for example, E. Meneghini et al., (1999) Nature 399: 793-797, show that the genes MOM-4 and LIT-1 were also required to down-regulate POP-1 not only in E but in other posterior daughter cells. MOM-4 and LIT-1 are homologous to the mammalian components of the mitogen-activated protein kinase (MAPK) pathway of TAK-1 (transforming growth factor beta activated kinase (and NLK) nemo-like kinase, respectively. MOM-4 and TAK-1 bind related proteins that promote their kinase activity. The authors of the report concluded that a MAPK-related pathway cooperates with Wnt signal transduction to down-regulate POP-1 activity.
In a further report by the same group (Ishitani et al, (1999) Nature 399: 798-802), it was shown that the TAK-1-NLK-MAPK-related pathway antagonizes signaling between beta-catenin and transcription factor TCF. The Wnt-signaling pathway regulates developmental processes through a complex of beta-catenin and the T-cell factor/lymphoid enhancer factor (TCF LEF) family of high-mobility group transcription factors. Wnt stabilizes beta-catenin which then binds to TCF and activates gene transcription. This signal pathway is conserved in vertebrates, Drosophilia and C. elegans. In C. elegans, MOM-4 and LIT-1 regulate Wnt signaling during embryogenesis. MOM-4 is homologous to TAK-1 (a kinase activated by transforming growth factor beta). LIT-1 is homologous to mitogen-activated protein kinase kinase kinase (MAP3K) and MAP kinase (MAPK)-related NEMO-like kinase (NLK) in mammalian cells. This raised the possibility that TAK-1 and NLK were involved in Wnt signaling in mammalian cells. The authors reported that TAK-1 activation stimulates NLK activity and down-regulates transcriptional activation mediated by beta-catenin and TCF. Injection of NLK suppressed the induction of axis duplication by microinjected beta-catenin in Xenopus embryos. NLK was shown to phosphorylate TCF LEF factors and inhibit the interaction of the beta-catenin-TCF complex with DNA. Accordingly, the TAK-1-NLK-MAPK-like pathway was shown to negatively regulate the Wnt signaling pathway.
Members of the tumor necrosis factor receptor superfamily have an important role in the induction of cellular signals resulting in cell growth, differentiation, and death. See Smith et al. (1994) Cell 76:959-962. Tumor necrosis factor receptor-1 recruits and assembles a signaling complex containing a number of death domain-containing proteins and a serine/threonine kinase, RIP, that mediates tumor necrosis factor-induced activation of nuclear factor-xcexaB. See Stanger et al. (1995) Cell 81:513-523 and Kelliher et al. (1998) Immunity 8:297-303. Recently, another RIP-like kinase has been characterized, designated xe2x80x9cCARDIAK,xe2x80x9d which contains a serine/threonine kinase domain as well as a carboxy-terminal caspase recruiting domain (CARD) (Thome, et al. (1998) Current Biology 8:885-888). Overexpression of this serine/threonine kinase induced the activation of both nuclear factor-xcexaB and Jun N-terminal kinase. This kinase also interacted with the tumor necrosis factor receptor-associated factors TRAF-1 and TRAF-2. A dominant negative form of TRAF-2 inhibited CARDIAK-induced nuclear factor-xcexaB activation. The data in the report suggested that CARDIAK is involved in nuclear factor-xcexaB/Jun N-terminal kinase signaling.
Protein kinases play critical roles in cellular growth. Therefore, novel protein kinase polynucleotides and proteins are useful for modulating cellular growth, differentiation and/or development.
In multicellular organisms, homeostasis is maintained by balancing the rate of cell proliferation against the rate of cell death. Cell proliferation is influenced by numerous growth factors and the expression of proto-oncogenes, which typically encourage progression through the cell cycle. In contrast, numerous events, including the expression of tumor suppressor genes, can lead to an arrest of cellular proliferation.
In differentiated cells, a particular type of cell death called apoptosis occurs when an internal suicide program is activated. This program can be initiated by a variety of external signals as well as signals that are generated within the cell in response to, for example, genetic damage. Dying cells are eliminated by phagocytes, without an inflammatory response.
Programmed cell death is a highly regulated process (Wilson (1998) Biochem. Cell. Biol. 76:573-582). The death signal is then transduced through various signaling pathways that converge on caspase-mediated degradative cascades resulting in the activation of late effectors of morphological and physiological aspects of apoptosis, including DNA fragmentation and cytoplasmic condensation. In addition, regulation of programmed cell death may be integrated with regulation of energy, redox- and ion homeostasis in the mitochondria (reviewed by (Kroemer, 1998)), and/or cell-cycle control in the nucleus and cytoplasm (reviewed by (Choisy-Rossi and Yonish-Rouach, 1998; Dang, 1999; Kasten and Giordano, 1998)). Many mammalian genes regulating apoptosis have been identified as homologs of genes originally identified genetically in Caenorhabditis elegans or Drosophila melanogaster, or as human oncogenes. Other programmed cell death genes have been found by domain homology to known motifs, such as death domains, that mediate protein-protein interactions within the programmed cell death pathway.
The mechanisms that mediate apoptosis include, but are not limited to, the activation of endogenous proteases, loss of mitochondrial function, and structural changes, such as disruption of the cytoskeleton, cell shrinkage, membrane blebbing, and nuclear condensation due to degradation of DNA. The various signals that trigger apoptosis may bring about these events by converging on a common cell death pathway that is regulated by the expression of genes that are highly conserved.
Caspases (cysteine proteases having specificity for aspartate at the substrate cleavage site) are central to the apoptotic program. These proteases are responsible for degradation of cellular proteins that lead to the morphological changes seen in cells undergoing apoptosis. One of the human caspases was previously known as the interleukin-1xcex2 (IL-1xcex2) converting enzyme (ICE), a cysteine protease responsible for the processing of pro-IL-1xcex2 to the active cytokine. Overexpression of ICE in Rat-1 fibroblasts induces apoptosis (Miura et al. (1993) Cell 75:653).
Many caspases and proteins that interact with caspases possess domains of about 60 amino acids called a caspase recruitment domain (CARD). Apoptotic proteins may bind to each other via their CARDs. Different subtypes of CARDs may confer binding specificity, regulating the activity of various caspases. (Hofmann et al. (1997) TIBS 22:155).
The functional significance of CARDs have been demonstrated in two recent publications. Duan et al. (1997) Nature 385:86 showed that deleting the CARD at the N-terminus of RAIDD, a newly identified protein involved in apoptosis, abolished the ability of RAIDD to bind to caspases. In addition, Li et al. (1997) Cell 91:479 showed that the N-terminal 97 amino acids of apoptotic protease activating factor-1 (Apaf-1) was sufficient to confer caspase-9-binding ability.
Thus, programmed cell death (apoptosis) is a normal physiological activity necessary to proper and differentiation in all vertebrates. Defects in apoptosis programs result in disorders including, but not limited to, neurodegenerative disorders, cancer, immunodeficiency, heart disease and autoimmune diseases (Thompson et al. (1995) Science 267:1456).
In vertebrate species, neuronal programmed cell death mechanisms have been associated with a variety of developmental roles, including the removal of neuronal precursors which fail to establish appropriate synaptic connections (Oppenheim et al. (1991) Annual Rev. Neuroscience 14:453-501), the quantitative matching of pre- and post-synaptic population sizes (Herrup et al. (1987) J. Neurosci. 7:829-836), and sculpting of neuronal circuits, both during development and in the adult (Bottjer et al. (1992) J. Neurobiol. 23:1172-1191).
Inappropriate apoptosis has been suggested to be involved in neuronal loss in various neurodegenerative diseases such as Alzheimer""s disease (Loo et al. (1993) Proc. Natl. Acad. Sci. 90:7951-7955), Huntington""s disease (Portera-Cailliau et al. (1995) J. Neurosc. 15:3775-3787), amyotrophic lateral sclerosis (Rabizadeh et al. (1995) Proc. Natl. Acad. Sci. 92:3024-3028), and spinal muscular atrophy (Roy et al. (1995) Cell 80:167-178).
In addition, improper expression of genes involved in apoptosis has been implicated in carcinogenesis. Thus, it has been shown that several xe2x80x9concogenesxe2x80x9d are in fact involved in apoptosis, such as in the Bcl family.
Accordingly, genes involved in apoptosis are important targets for therapeutic intervention. It is important, therefore, to identify novel genes involved in apoptosis or to discover whether known genes function in this process.
An isolated nucleic acid molecule corresponding to a protein kinase nucleic acid sequence is provided. Additionally an amino acid sequence corresponding to the polynucleotide is encompassed. In particular, the present invention provides for an isolated nucleic acid molecule (SEQ ID NO:1) comprising the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2. Further provided is a protein kinase polypeptide having an amino acid sequence encoded by the nucleic acid molecule described herein. The coding sequence for human 14171 is shown in SEQ ID NO:3.
The present invention also provides vectors and host cells for recombinant expression of the nucleic acid molecule described herein, as well as methods of making such vectors and host cells and for using them for production of the polypeptide or peptides of the invention by recombinant techniques.
The protein kinase molecule of the present invention is useful for modulating cellular growth and/or cellular metabolic pathways particularly for regulating one or more proteins involved in growth and metabolism. Accordingly, in one aspect, this invention provides an isolated nucleic acid molecule encoding a protein kinase protein or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of protein kinase-encoding nucleic acids.
Another aspect of this invention features an isolated or recombinant kinase protein and polypeptide. Preferred protein kinase proteins and polypeptides possess at least one biological activity possessed by naturally-occurring protein kinase.
Variant nucleic acid molecules and polypeptides substantially homologous to the nucleotide and amino acid sequence set forth in the sequence listing are encompassed by the present invention. Additionally, fragments and substantially homologous fragments of the nucleotide and amino acid sequence are provided.
Antibodies and antibody fragments that selectively bind the protein kinase polypeptide and fragments are provided. Such antibodies are useful in detecting the protein kinase polypeptide as well as in modulating cellular growth and metabolism.
In another aspect, the present invention provides a method for detecting the presence of protein kinase activity or expression in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of protein kinase activity such that the presence of protein kinase activity is detected in the biological sample.
In yet another aspect, the invention provides a method for modulating protein kinase activity comprising contacting a cell with an agent that modulates (inhibits or stimulates) protein kinase activity or expression such that protein kinase activity or expression in the cell is modulated. In one embodiment, the agent is an antibody that specifically binds to kinase protein. In another embodiment, the agent modulates expression of protein kinase protein by modulating transcription of a protein kinase gene, splicing of a protein kinase mRNA, or translation of a protein kinase mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of the protein kinase mRNA or the protein kinase gene.
In one embodiment, the methods of the present invention are used to treat a subject having a disorder characterized by aberrant protein kinase protein activity or nucleic acid expression by administering an agent that is a protein kinase modulator to the subject. In one embodiment, the protein kinase modulator is a protein kinase protein. In another embodiment, the protein kinase modulator is a protein kinase nucleic acid molecule. In other embodiments, the protein kinase modulator is a peptide, peptidomimetic, or other small molecule.
The present invention also provides a diagnostic assay for identifying the presence or absence of a genetic lesion or mutation characterized by at least one of the following: (1) aberrant modification or mutation of a gene encoding a protein kinase protein; (2) misregulation of a gene encoding a protein kinase protein; and (3) aberrant post-translational modification of a protein kinase protein, wherein a wild-type form of the gene encodes a protein with a protein kinase activity.
In another aspect, the invention provides a method for identifying a compound that binds to or modulates the activity of a protein kinase protein. In general, such methods entail measuring a biological activity of a protein kinase protein in the presence and absence of a test compound and identifying those compounds that alter the activity of the protein kinase protein.
The invention also features methods for identifying a compound that modulates the expression of the protein kinase gene by measuring the expression of the protein kinase sequence in the presence and absence of the compound.
The invention also provides compounds identified by the screening methods described herein.
Other features and advantages of the invention will be apparent from the following detailed description and claims.